RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets
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PROVIDER: PRJNA431613 | ENA |
REPOSITORIES: ENA
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Project description:MicroRNAs are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC complex functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibody and compiled a dataset made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on predicted miRNA binding sites, both in the 3’UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the differentially expressed genes, independently for AGO2 and GW182 related samples. For each of the two proteins, we trained and tested several support vector machine algorithms able to predict the differentially expressed genes that were experimentally detected. The most efficient algorithm for predicting the over-expressed genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3’UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for predicting over-expressed genes in the GW182 immunoprecipitated sample was the length of the coding region. Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC complex recruits genes based on miRNA binding sites in the 3’UTR and coding region, but only the longer mRNAs remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.
2018-09-05 | GSE109666 | GEO
Project description:MicroRNAs are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC complex functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibody and compiled a dataset made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on predicted miRNA binding sites, both in the 3’UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the differentially expressed genes, independently for AGO2 and GW182 related samples. For each of the two proteins, we trained and tested several support vector machine algorithms able to predict the differentially expressed genes that were experimentally detected. The most efficient algorithm for predicting the over-expressed genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3’UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for predicting over-expressed genes in the GW182 immunoprecipitated sample was the length of the coding region. Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC complex recruits genes based on miRNA binding sites in the 3’UTR and coding region, but only the longer mRNAs remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.
2018-09-05 | GSE109665 | GEO
Project description:RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets (mRNA)
| PRJNA431618 | ENA
Project description:RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets (microRNA)
| PRJNA431617 | ENA
Project description:BackgroundMicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line.MethodsWe performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on miRNA binding sites, both in the 3'UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the enriched or underrepresented genes in the immunoprecipitated fractions, independently for AGO2 and GW182 related samples.ResultsFor each of the two proteins, we trained and tested several support vector machine algorithms capable of distinguishing the enriched from the underrepresented genes that were experimentally detected. The most efficient algorithm for distinguishing the enriched genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3'UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for distinguishing the enriched genes in the GW182 immunoprecipitated samples was the length of the coding region.ConclusionsDue to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC recruits genes based on miRNA binding sites in the 3'UTR and coding region, but only the longer mRNAs probably remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.
| S-EPMC6471694 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2018-09-05 | GSE109667 | GEO
Project description:To determine the spectrum of miRNA targets regulated following Dicer deletion, we performed argonaute 2 (AGO2)-RNA Immunoprecipitation (RIP)-microarray in bone marrow-derived macrophages (BMDMs) from LysM-Cre/Dicerflox/flox/Apoe–/– and LysM-Cre/Dicerwt/wt/Apoe–/– mice. This analysis combined with miRNA profiling in Dicer wild type (WT) and knockout (KO) BMDMs may help to identify the miRNA targets regulated by Dicer deletion.
2018-05-23 | GSE87717 | GEO
Project description:We undertook an unbiased approach based on differential Argonaute-2 (Ago2) RNA immunoprecipitation followed by deep sequencing (Ago2 RIP-seq) to unravel the molecular mechanism(s) underlying miR-146a function, namely the relevant mRNA target(s). Ago2 is a crucial protein that engages with both miRNA and its mRNA target(s) in the RISC complex. To overcome the problem of lack of commercially available high affinity antibodies against Ago2, we took advantage of a genetically modified mouse in which endogenous Ago2 is replaced with a N-terminal tagged version of the protein (3xFLAG- 6xHIS), thus allowing stringent purification (i.e., with low unspecific background) of the RNA molecules bound to Ago2 (upon UV-crosslinking), followed by RNA-sequencing. In order to enrich for miR-146a targets, we transduced purified CD3+ T cells (we could not use γδ T cells due to insufficient cell numbers for this approach) with a miR-146a-expressing retrovirus (with~12-fold overexpression of miR-146a). We thereby identified 225 putative mRNAs after differential Ago2-RIP-seq, from which 96 (42,7%) had a predicted miR-146a binding site in their 3’ UTR region.
2018-02-12 | GSE109263 | GEO
Project description:RNA immunoprecipitation sequencing (RIP seq) using anti-Argonaute2 (Ago2) antibody to identify the potential targets of kshv-miR-K12-1-5p in AC16 cells.
2022-04-22 | GSE201041 | GEO
Project description:Shortening of polyadenosine (poly(A)) tails (deadenylation) is the initial step in the decay of most mRNAs. The CCR4-NOT complex is the major deadenylase in mammals. Interaction of Cnot1 with RNA binding proteins, including the miRNA-induced silencing complex (miRISC), which includes Argonaute (Ago) and GW182 as core proteins, and the TTP family of AU-rich element (ARE)-binding proteins (TTP and BRF1/2), recruits the CCR4-NOT complex to the 3’-untranslated regions (3’-UTR) of mRNAs, leading to their degradation. This experiment aimed to determine the binding affinity of the CCR4-NOT complex, Ago2, and BRF1 with their target mRNAs in liver. Liver isolated from C57BL/6J wild-type mice at 8 weeks of age was solubilized in TNE buffer for 30 min at 4ºC. Lysate was incubated with antibodies against Cnot3, Ago2, and BRF1 for 1 hr at 4ºC, and then incubated with Dynabeads (Invitrogen) for 2 hr at 4ºC. mRNAs in immune complexes were isolated using Isogen II. 100 ng of total RNA was used for RNA-seq library preparation with TruSeq Stranded mRNA Library Prep Kit for NeoPrep. 150 base-pair pair-end read RNA-seq was performed with Hiseq 3000/4000 PE Cluster Kit and Hiseq 3000/4000 SBS Kit on Hiseq4000.
2020-03-30 | E-MTAB-6941 | biostudies-arrayexpress